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Backhousia citriodora (lemon myrtle) extract has been found to inhibit glucansucrase activity, which plays an important role in biofilm formation by Streptococcus mutans. In addition to glucansucrase, various virulence factors in S. mutans are involved in the initiation of caries. Lactate produced by S. mutans demineralizes the tooth enamel. This study investigated whether lemon myrtle extract can inhibit S. mutans lactate production. Lemon myrtle extract reduced the glycolytic pH drop in S. mutans culture and inhibited lactate production by at least 46%. Ellagic acid, quercetin, hesperetin, and myricetin, major polyphenols in lemon myrtle, reduced the glycolytic pH drop and lactate production, but not lactate dehydrogenase activity. Furthermore, these polyphenols reduced the viable S. mutans cell count. Thus, lemon myrtle extracts may inhibit S. mutans-mediated acidification of the oral cavity, thereby preventing dental caries and tooth decay., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Herein, we investigated enzymatic properties and reaction specificities of Streptococcus mutans dextranase, which hydrolyzes α-(1→6)-glucosidic linkages in dextran to produce isomaltooligosaccharides. Reaction specificities of wild-type dextranase and its mutant derivatives were examined using dextran and a series of enzymatically prepared p-nitrophenyl α-isomaltooligosaccharides. In experiments with 4-mg·mL -1 dextran, isomaltooligosaccharides with degrees of polymerization (DP) of 3 and 4 were present at the beginning of the reaction, and glucose and isomaltose were produced by the end of the reaction. Increased concentrations of the substrate dextran (40 mg·mL -1 ) yielded isomaltooligosaccharides with higher DP, and the mutations T558H, W279A/T563N, and W279F/T563N at the -3 and -4 subsites affected hydrolytic activities of the enzyme, likely reflecting decreases in substrate affinity at the -4 subsite. In particular, T558H increased the proportion of isomaltooligosaccharide with DP of 5 in hydrolysates following reactions with 4-mg·mL -1 dextran.Abbreviations CI: cycloisomaltooligosaccharide; CITase: CI glucanotransferase; CITase-Bc: CITase from Bacillus circulans T-3040; DP: degree of polymerization of glucose unit; GH: glycoside hydrolase family; GTF: glucansucrase; HPAEC-PAD: high performance anion-exchange chromatography-pulsed amperometric detection; IG: isomaltooligosaccharide; IGn: IG with DP of n (n, 2‒5); PNP: p-nitrophenol; PNP-Glc: p-nitrophenyl α-glucoside; PNP-IG: p-nitrophenyl isomaltooligosaccharide; PNP-IGn: PNP-IG with DP of n (n, 2‒6); SmDex: dextranase from Streptococcus mutans; SmDexTM: S. mutans ATCC25175 SmDex bearing Gln100‒Ile732.

Genes encoding fluoride transporters have been identified in bacterial and archaeal species. The genome sequence of the cariogenic Streptococcus mutans bacteria suggests the presence of a putative fluoride transporter, which is referred to as a chloride channel permease. Two homologues of this gene (GenBank locus tags SMU_1290c and SMU_1289c) reside in tandem in the genome of S. mutans The aim of this study was to determine whether the chloride channel permeases contribute to fluoride resistance. We constructed SMU_1290c- and SMU_1289c-knockout S. mutans UA159 strains. We also constructed a double-knockout strain lacking both genes. SMU_1290c or SMU_1289c was transformed into a fluoride transporter- disrupted Escherichia coli strain. All bacterial strains were cultured under appropriate conditions with or without sodium fluoride, and fluoride resistance was evaluated. All three gene-knockout S. mutans strains showed lower resistance to sodium fluoride than did the wild-type strain. No significant changes in resistance to other sodium halides were recognized between the wild-type and double-knockout strains. Both SMU_1290c and SMU_1289c transformation rescued fluoride transporter-disrupted E. coli cell from fluoride toxicity. We conclude that the chloride channel permeases contribute to fluoride resistance in S. mutans., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Streptococcus mutans harbours an intracellular, human DPP IV-analogous enzyme Xaa-Pro dipeptidyl-peptidase (EC 3.4.14.11). According to previous reports, an extracellular isozyme in S. gordonii and S. suis has been associated with virulence. Speculating that even an intracellular form may aid in virulence of S. mutans, we have tried to purify, characterize and evaluate enzyme inhibition by specific inhibitors. The native enzyme was partially purified by ion-exchange and gel filtration chromatography. Owing to low yield, the enzyme was overexpressed in Lactococcus lactis and purified by affinity chromatography. The recombinant enzyme (rSm-XPDAP) had a specific activity of 1070 U mg(-1), while the Vmax and Km were 7 μM min(-1) and 89 ± 7 μM (n = 3), respectively. The serine protease inhibitor phenylmethylsulphonyl fluoride and a DPP IV-specific inhibitor diprotin A proved to be active against rSm-XPDAP. As a novel approach, the evaluation of the effect of anti-human DPP IV (AHD) drugs on rSm-XPDAP activity found saxagliptin to be effective to some extent (Ki = 129 ± 16 μM), which may lead to the synthesis and development of a new class of antimicrobial agents., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The 23S rRNA nucleotide m(2)G2445 is highly conserved in bacteria, and in Escherichia coli this modification is added by the enzyme YcbY. With lengths of around 700 amino acids, YcbY orthologs are the largest rRNA methyltransferases identified in Gram-negative bacteria, and they appear to be fusions from two separate proteins found in Gram-positives. The crystal structures described here show that both the N- and C-terminal halves of E. coli YcbY have a methyltransferase active site and their folding patterns respectively resemble the Streptococcus mutans proteins Smu472 and Smu776. Mass spectrometric analyses of 23S rRNAs showed that the N-terminal region of YcbY and Smu472 are functionally equivalent and add the m(2)G2445 modification, while the C-terminal region of YcbY is responsible for the m(7)G2069 methylation on the opposite side of the same helix (H74). Smu776 does not target G2069, and this nucleotide remains unmodified in Gram-positive rRNAs. The E.coli YcbY enzyme is the first example of a methyltransferase catalyzing two mechanistically different types of RNA modification, and has been renamed as the Ribosomal large subunit methyltransferase, RlmKL. Our structural and functional data provide insights into how this bifunctional enzyme evolved.

Dextran glucosidase from Streptococcus mutans (SmDG), which belongs to glycoside hydrolase family 13 (GH13), hydrolyzes the non-reducing terminal glucosidic linkage of isomaltooligosaccharides and dextran. Thermal deactivation of SmDG did not follow the single exponential decay but rather the two-step irreversible deactivation model, which involves an active intermediate having 39% specific activity. The presence of a low concentration of CaCl2 increased the thermostability of SmDG, mainly due to a marked reduction in the rate constant of deactivation of the intermediate. The addition of MgCl2 also enhanced thermostability, while KCl and NaCl were not effective. Therefore, divalent cations, particularly Ca2+, were considered to stabilize SmDG. On the other hand, CaCl2 had no significant effect on catalytic reaction. The enhanced stability by Ca2+ was probably related to calcium binding in the β→α loop 1 of the (β/α)(8) barrel of SmDG. Because similar structures and sequences are widespread in GH13, these GH13 enzymes might have been stabilized by calcium ions.

Streptococcus mutans, an etiological agent of dental caries, is a normal inhabitant of dental plaque. Two main virulence factors of S. mutans are acidogenicity and aciduricity - the ability to produce acid and survive at low pH, respectively. Metabolic processes, including the catabolism of pyruvate, are finely regulated following acid exposure in S. mutans. Proteome analysis of the S. mutans acid response has shown pyruvate dehydrogenase A (PdhA) is upregulated. PdhA is the E1alphasubunit of the four-enzyme pyruvate dehydrogenase complex, responsible for the heterofermentative catalysis of pyruvate into acetyl-CoA. Acetyl-CoA is subsequently catalyzed into ethanol and acetate yielding additional ATP. This investigation examined the relationships between PdhA, aciduricity, and metabolism in S. mutans. An S. mutans pdhA knockout (PDHAKO) revealed an acid sensitive phenotype, displayed by increased doubling times, and decreased competitiveness in a biofermentor. Quantitative real-time PCR showed pdhA expression increased dramatically during acidic growth and under acid adaptation. Additionally, pdhA expression responded to conditions favoring heterofermentative growth; decreased in the presence of excess glucose, and increased during stationary phase compared with mid-log phase growth. This study demonstrated that in S. mutans, pdhA expression responds to conditions conducive to heterofermentation and deletion of pdhA resulted in decreased aciduricity.

The aim of this study was to examine the effects of 7-epiclusianone, a new prenylated benzophenone isolated from the plant Rheedia gardneriana, on some of the virulence properties of Streptococcus mutans associated with biofilm development and acidogenicity. The synthesis of glucans by glucosyltransferases B (GTF B) and C (GTF C) was markedly reduced by 7-epiclusianone showing more than 80% inhibition of enzymatic activity at a concentration of 100 microg mL(-1). Double-reciprocal analysis (Lineweaver-Burk plots) revealed that the inhibition of GTF B activity was noncompetitive (mixed) while GTF C was inhibited uncompetitively. The glycolytic pH drop by S. mutans cells was also disrupted by 7-epiclusianone without affecting the bacterial viability, an effect that can be attributed, in part, to inhibition of F-ATPase activity (61.1+/-3.0% inhibition at 100 microg mL(-1)). Furthermore, topical applications (1-min exposure, twice daily) of 7-epiclusianone (at 250 microg mL(-1)) disrupted biofilm formation and physiology. The biomass (dry-weight), extracellular insoluble polysaccharide concentration and acidogenicity of the biofilms were significantly reduced by the test agent (P<0.05). The data show that 7-epiclusianone disrupts the extracellular and intracellular sugar metabolism of S. mutans, and holds promise as a novel, naturally occurring compound to prevent biofilm-related oral diseases.

Transglucosylation from sucrose to acetic acid by sucrose phosphorylase (EC 2.4.1.7) was studied. 1-O-Acetyl-alpha-D-glucopyranose was isolated as the main product of the enzyme reaction. We also compared the pH-dependence of transglycosylation catalyzed by sucrose phosphorylase toward carboxyl and hydroxyl groups. With hydroquinone as an acceptor molecule, the transfer ratio of glucose residue was higher at neutral pH. This pH-activity profile was similar to that of the phosphorolysis of sucrose by sucrose phosphorylase, but with acetic acid as an acceptor molecule, the transfer ratio of glucose residue was higher at low pH. These findings suggest that the undissociated carboxyl group is essential to the acceptor molecule for the transglycosylation reaction of sucrose phosphorylase. In a sensory test, the sour taste of acetic acid was markedly reduced by glucosylation. The threshold value of the sour taste of acetic acid glucosides was approximately 100 times greater than that of acetic acid.

Streptococcus mutans and certain other oral lactic-acid bacteria were found to have the ability to carry out malolactic fermentation involving decarboxylation of L-malate to yield L-lactic acid and concomitant reduction in acidity. The activity was inducible by L-malate in S. mutans UA159 growing in suspensions or biofilms. The optimal pH for the fermentation was c. 4.0 for both suspensions and biofilms, although the pH optimum for malolactic enzyme in permeabilized cells of S. mutans UA159 was close to 5.5. Although malate did not serve as a catabolite for growth of S. mutans, it did serve to protect the organism against acid killing and to maintain ATP pool levels during starvation. Alkalinization associated with malolactic fermentation resulted in pH rise or increased need to add standardized HCl solution to maintain a set pH value in pH-stat experiments. The net conclusion is that malate has the potential to be effective for alkalinization of dental plaque, although the fermentation is sensitive to fluoride and triclosan, which are commonly added to oral care products.

We examined the effects of phosphorothioate-modified antisense oligodeoxyribonucleotides (PS-ODN) targeted to mRNA transcribed from gtfB, which encodes synthesis of water-insoluble glucans in Streptococcus mutans. Treatment of S. mutans with 10 muM antisense PS-ODNs inhibited gtfB mRNA transcription, GtfB expression and water-insoluble glucan synthesis. The architecture of biofilms formed by antisense PS-ODNs-treated S. mutans showed reduced biomass, more dispersed distribution with enlarged interspaces and fewer layers of attached cells. PS-ODN treatment had no effect on the growth of S. mutans. Our results indicated that it might be feasible to use antisense PS-ODN as a novel agent in caries prevention.

Objectives: The glucosyltransferase (GTF) and fructosyltransferase (FTF) enzymes play a pivotal role in dental biofilm formation as they synthesize polysaccharides that act as the extracellular matrix of the biofilm. Iodine is a unique antibacterial agent that has distinct properties from other conventional antibacterial agents. In this study we have examined the effect of iodine and povidone iodine (PI) on gtf and ftf expression in biofilm and planktonic environments and on immobilized and unbound GTF and FTF activity., Methods: Real-time reverse transcription-PCR was used to investigate the effect of iodine and PI on ftf, gtfB and gtfC expression. The effect of iodine and PI on GTF and FTF activity was tested using radioactive assays., Results: Our results indicate that iodine and PI in a tetraglycol carrier cause enhancement of expression of gtfB in Streptococcus mutans in biofilms but not in planktonic bacteria. PI in water induced expression of gtfB and gtfC in planktonic bacteria. However, iodine and PI strongly inhibit polysaccharide production by GTF and to a lesser extent by FTF activity. The inhibitory effect on GTF activity was similar in solution compared to its activity in the immobilized environment. This unique effect may be attributed to the distinct chemical properties of iodine compared with other antibacterial agents., Conclusions: This study indicates that iodine at sub-bactericidal concentrations demonstrates molecular and enzymatic effects that are highly associated with biofilm formation.

Fluoride and zinc, alone or in combination at concentrations of 0.2 mM, inhibited production-secretion of glucosyltranferases by Streptococcus mutans UA159 growing in suspension cultures. Inhibition did not involve growth inhibition or starvation. Fluoride and zinc also inhibited glucan production, especially insoluble glucan, in fed-batch biofilms. Inhibition of biofilms appeared to be associated with starvation as indicated by markedly decreased ATP pools and iodophilic polysaccharide levels in biofilm cells. As insoluble glucans are important for virulence of mutans streptococci, the inhibitory actions of fluoride and zinc could significantly affect cariogenicity.

The glucan that was produced by glucosyltransferases (GTFs) from Streptococcus mutans was examined for its stimulating functions toward murine peritoneal macrophages. Soluble glucan was obtained by the reaction with cell-free crude GTFs and sucrose, followed by ethanol precipitation, dispersion in water and re-precipitation by ethanol. Soluble glucan, those average molecular weight was about 3 x 10(5), was composed of mixture of alpha-1,6 and alpha-1,3 linkages in a 3:1 ratio. When 30 and 60 microg/ml of the glucan was incubated with peritoneal macrophages, the lysosomal phosphatase activity was increased in a dose-dependent manner, indicating that soluble glucan may activate macrophages. To examine its effects on the various functions of macrophages, soluble glucan was orally administered daily at a level of 100 mg/kg of body weight to C57BL/6 mice. Significant stimulation of the production of H2O2 by the macrophages was observed without any increase in NO production. The production of tumor necrosis factor-alpha (TNF-alpha) by the macrophages was also stimulated from 538.73-555.06 pg/ml to 585.73-596.40 pg/ml during 15 days of oral administration of soluble glucan. The cytotoxicity of peritoneal macrophages against B16 tumor cells was significantly enhanced by 25-38% during 15 days of oral administration. These results may indicate that soluble glucan stimulates the immune functions of macrophages.

Glucosyltransferases (Gtfs) and fructosyltransferase (Ftf), and the exopolysaccharides they produce, facilitate bacterial adherence and biofilm formation, and enhance the virulence of Streptococcus mutans. In this study, we used continuous chemostat cultures and reporter gene fusions to study the expression of ftf and gtfBC in response to carbohydrate availability and pH, and to asses the role of a protein similar to catabolite control protein A (CcpA), RegM, in regulation of these genes. Expression of ftf was efficient at pH 7.0 and 6.0, but was repressed at pH 5.0 under glucose-excess conditions. At pH 7.0, ftf expression was 5-fold lower under glucose-limiting conditions than in cells growing with an excess of glucose. Expression of gtfBC was also sensitive, albeit to a lesser extent, to pH and glucose availability. Inactivation of regM resulted in decreases of as much as 10-fold in both ftf and gtfBC expression, depending on growth conditions. These findings reinforce the importance of pH and carbohydrate availability for expression of two primary virulence attributes of S. mutans and reveal a critical role for RegM in regulation of expression of both gtfBC and ftf.

Previously, we described in Streptococcus mutans strain NG8 a 5-gene operon (sat) that includes ffh, the bacterial homologue of the eukaryotic signal recognition particle (SRP) protein, SR54. A mutation in ffh resulted in acid sensitivity but not loss of viability. In the present study, chemostat-grown cells of the ffh mutant were shown to possess only 26% and 39% of the parental membrane F-ATPase activity and 55% and 75% of parental glucose-phosphotransferase (PTS) activity when pH-7 and pH-5-grown cells, respectively, were assayed. Two-dimensional-gel electrophoretic analyses revealed significant differences in protein profiles between parent and ffh-mutant strains at both pH 5 and pH 7. It appears that the loss of active SRP (Ffh) function, while not lethal, results in substantial alterations in cellular physiology that includes acid tolerance., (Copyright 2004 Federation of European Microbiological Societies)

Streptococcus mutans utilizes sucrose to synthesize glucans by glucosyltransferase and fructans by fructosyltransferase (FTF). Antibodies raised against a recombinant FTF were used to study S. mutans FTF secretion. Low amounts of cell-free FTF were found in culture of S. mutans grown with sucrose, while an increase in bacteria displaying cell surface FTF was detected. FTF added to S. mutans cultures was adsorbed to bacteria grown with sucrose but not to bacteria grown with glucose or fructose or to a gtf inactivated mutant grown with sucrose. Recombinant FTF was found to have high affinity for glucans suggesting that fructans and glucans are an integral part of the polysaccharide matrix of oral biofilms.

We isolated a spontaneous suppressor mutant complementing the acid-sensitive phenotype of Streptococcus mutans strain Tn-1, a mutant previously generated in this laboratory, defective in the activity of the dgk-encoded putative undecaprenol kinase. A relatively simple genetic method was developed to identify the suppressor mutation, based on selection for transformants containing two closely linked markers: a selectable allele of the unknown suppressor gene and an antibiotic resistance gene introduced on a suicide plasmid at random sites into the chromosome via homologous recombination. While we have not actually identified the original suppressor mutation, another mutated gene restoring acid resistance has been isolated, which suggests a possible mechanism of suppression.

An oolong tea polyphenol (OTF6) has been shown to possess a strong anti-glucosyltransferase (GTF) activity and inhibit experimental dental caries in rats infected with mutans streptococci. The effects of OTF6 on the functional domains of GTFs of Streptococcus mutans, an N-terminal catalytic domain (CAT), and a C-terminal glucan-binding domain (GBD), were examined. The maximum velocity of glucan synthesis by recombinant GTFB (rGTFB) and GTFD (rGTFD) became significantly slower in the presence of OTF6, however, Km values remained stable when compared in their absence. These results suggest that OTF6 reduces glucan synthesis by non-competitively inhibiting the GBD of S. mutans GTFB and GTFD. Further, the recombinant proteins of CAT (rCAT) and GBD (rGBD) were expressed using Escherichia coli, and purified by affinity column chromatography. rGBD but not rCAT was found to possess dextran-binding activity, which was shown to be inhibited by OTF6. These results indicate that OTF6, a polymeric polyphenol specific for oolong tea is able to reduce glucan synthesis by inhibiting the GBD of S. mutans GTFB.

Aims: To examine the dextran-binding domain of the dextranase (Dex) of Streptococcus mutans., Methods and Results: Deletion mutants of the Dex gene of Strep. mutans were prepared by polymerase chain reaction and expressed in Escherichia coli cells. Binding of the truncated Dexs to dextran was measured with a Sephadex G-150 gel. Although the Dexs which lacked the N-terminal variable region lost enzyme activity, they still retained dextran-binding ability. In addition, further deletion into the conserved region from the N-terminal did not influence the dextran-binding ability. However, the Dex which carried a deletion in the C-terminus still possessed both enzyme activity and dextran-binding ability. Further deletion into the conserved region from the C-terminal resulted in complete disappearance of both enzyme and dextran-binding activities., Conclusions: Deletion analysis of the Dex gene of Strep. mutans showed that the C-terminal side (about 120 amino acid residues) of the conserved region of the Dex was essential for dextran-binding ability., Significance and Impact of the Study: The dextran-binding domain was present in a different area from the catalytic site in the conserved region of the Dex molecule. The amino acid sequence of the dextran-binding domain of the Dex differed from those of glucan-binding regions of other glucan-binding proteins reported.

Aims: To investigate the formation of fructosyltransferase (FTF) complexes on hydroxyapatite (HA) surfaces., Methods and Results: Cell-free extracellular FTF from Streptococcus mutans, purified from hyperproducing strain V-1995, was adsorbed onto HA and then eluted from the surface by means of a concentration gradient of potassium phosphate buffer. The FTF monomers loaded onto HA formed, upon adsorption, various complexes ranging from 200 to 700 kDa as demonstrated using native PAGE. All these complexes exhibited enzymatic activity., Conclusions: Adsorption of FTF onto HA induced the formation of stable and enzymatically-active complexes., Significance and Impact of the Study: The formation of these complexes may explain the change of FTF catalytic properties after adsorption onto HA. This study is another step in determining the properties of a-cellular constituents of the oral biofilm.

Three genes, designated as fruC, fruD and fruI, were predicted to encode polypeptides homologous to fructose-specific enzyme II (II(Fru)) of the phosphoenolpyruvate-dependent sugar:phosphotransferase system, and were cloned from Streptococcus mutans, the primary etiological agent of human dental caries. The fruC and fruD genes encoded domains BC and domain A of II(Fru), respectively. The fruI gene encoded IICBA(Fru). Northern hybridization and slot blot analysis showed that expression of fruI was inducible by sucrose and fructose, while fruCD were expressed constitutively and at much lower levels. Inactivation of either fruI or fruCD alone, or of both fruCD and fruI, had no major impact on growth on fructose at a concentration of 0.5% (w/v). However, when the strains were grown with 0.2% fructose as the sole carbohydrate source, a significant decrease in the growth rate was seen with the fruCD/fruI double mutants. Assays of sugar:phosphotransferase activity showed that the fruCD/fruI double mutants had roughly 30% of the capacity of the wild-type strain to transport fructose via the phosphoenolpyruvate-dependent sugar:phosphotransferase system. Xylitol toxicity assays indicated that the inducible fructose permease was responsible for xylitol transport.

A homologue of the HtrA family of stress-response proteases was detected by analysis of the Streptococcus mutans genome sequence. Disabling of the S. mutans htrA gene by insertional inactivation resulted in bacterial clumping in liquid medium, altered colony morphology and a reduced ability to withstand high temperature, extremes of pH or oxidative stress. Seven different extracellular or wall-associated proteins that are known to be subject to post-translational proteolysis were examined in cultures of wild-type S. mutans and an htrA mutant. Inactivation of the htrA protease had no effect on degradation of the proteins.

Glucosyltransferases (GtfB/C/D) of Streptococcus mutans, a pathogen for human dental caries, synthesize water-insoluble glucan through the hydrolysis of sucrose. Genetic and biochemical approaches have identified several active sites of these enzymes, but no three-dimensional (3D) structural evidence is yet available to elucidate the subdomain arrangement and molecular mechanism of catalysis. Based on a combined sequence and secondary structure alignment against known crystal structures of segments from closely related proteins, we propose here the 3D model of an N-terminal domain essential for the sucrose binding and splitting in GtfB. A Tim-barrel of (alpha/beta)(8) structural characteristics is revealed and the structural correlation for two peptides is described.

Alkyl hydroperoxide reductase in Streptococcus mutans consists of two components, Nox-1 and AhpC. Deletion of nox-1 and ahpC in a double mutant as well as the wild-type of Streptococcus mutans can form colonies in the presence of air to the same extent. The evidence suggested the presence of some other antioxidant system(s) independent of the Nox-1/AhpC system in the bacterium. Here we identified a new antioxidant gene (dpr) and the gene product (Dpr) which complements the defect of peroxidase activity caused by the deletion of nox-1 and ahpC in S. mutans. The dpr-disruption mutant of S. mutans could form colonies anaerobically but not aerobically.

The role of F1F0-ATPase in Streptococcus mutans GS5 was investigated by isolating a mutant (NTS1) defective in enzyme activity by homologous recombination with a plasmid encoding the 5' terminal fragment of the F1F0-ATPase beta-subunit gene. The ATPase activity of NTS1 membranes was 49% that of GS5 membranes. The lag phase of the growth curve of NTS1 was longer than that for GS5, and the lag phase of GS5 and NTS1 were prolonged by the addition of ionophore gramicidin D; at stationary phase, the turbidity of the NTS1 culture was less than that of the GS5 culture. These results suggest that S. mutans F1F0-ATPase contributes to the generation of a stoichiometric electrogenic gradient effectively in the lag phase.

We cloned and sequenced the glutathione reductase gene (gor) of an oxygen-tolerant Streptococcus mutans, and constructed a gor-disruption mutant by homologous recombination. The gor gene consisted of 1,350 bp, coding for a protein of 450 amino acid residues. The deduced amino acid sequence of the S. mutans gor gene product showed extensive similarity with those of glutathione reductases from prokaryotes and eukaryotes. Although the mutant could grow aerobically, it showed no growth in the presence of 2 mM diamide, a thiol-specific oxidant. In contrast, growth of the wild-type strain was not significantly inhibited by 2 mM diamide, and glutathione reductase activity was increased 2.2-fold under these conditions. In addition, the level of glutathione reductase activity in the wild-type strain was increased 3.6-fold upon exposure to air, and the elevated level of the enzyme was retained throughout the aerobic growth. Thus, glutathione reductase may be important in protection of S. mutans against oxidative stress.

Three glucosyltransferase (GTase) genes (gtfB, gtfC and gtfD) were cloned and sequenced from clinically isolated strains of Streptococcus mutans MT8148 (serotype c), MT4239 (c), MT4245 (e), MT4467 (e) and MT4251 (f), respectively. Comparison of the gtf genes revealed that interstrain difference of gtfB and gtfD was limited, while gtfC showed significant interstrain variations. Similar to gtfB and gtfD, gtfC possessed five direct repeats composed of homologous unit in the carboxyl-terminal portion. The repeating unit consisted of 63-65 amino acid residues and is responsible for glucan binding. The gtfC gene from S. mutans MT4245 lacked the fourth unit. Multiple alignment with the gtf sequence of strain GS-5 (c) revealed several changes in these gtf genes due to frameshift mutations. The peptides encoded by the gtfB, gtfC and gtfD genes of GS-5 were 1, 80, and 32 amino acid residues shorter than those of the test strains except strain MT4245.

Oral streptococci are sugar-fermentative bacteria comprising at least 19 distinct species and are a significant proportion of the normal microbial population of the mouth and upper respiratory tract of humans. These streptococci transport several sugars by the phosphoenolpyruvate:sugar phosphotransferase system (PTS) which concomitantly catalyzes the phosphorylation and translocation of mono- and disaccharides via a chain of enzymic reactions that transfer a phosphate group from phosphoenolpyruvate to the incoming sugar. A number of PTS components, including HPr, Enzyme I and some Enzymes II, have been studied at the biochemical and/or genetical level in Streptococcus salivarius, Streptococcus mutans and Streptococcus sobrinus. Moreover, compelling evidence indicates that the oral streptococcal PTS is involved in the regulation of sugar metabolism. Results are accumulating suggesting that a protein called IIABMan, as well as the phosphocarrier protein HPr, are key regulatory components that allow these bacteria to select rapidly metabolizable sugars, such as glucose or fructose, over less readily utilizable carbohydrates. Circumstantial evidence suggests that the molecular mechanisms by which oral streptococcal PTS exert their regulatory functions differ from mechanisms in other Gram-negative or Gram-positive bacteria.

We previously established murine hybridomas producing a monoclonal antibody monospecific against three glucosyltransferases (I, SI and S) of Streptococcus mutans which contribute to dental caries formation. Here, we developed a new immunochemical technique (cross-dot system) with which individual levels of glucosyltransferases expressed by S. mutans can be evaluated. We also examined glucosyltransferase production and in vitro artificial plaque formation by a reference strain and several clinical isolates of S. mutans. The findings indicate that the levels of glucosyltransferases produced greatly vary with the cells and the culture medium, and that the cells producing high levels of both glucosyltransferase-SI and glucosyltransferase-I enzymes may possess high in vitro artificial plaque forming ability. We suggest that the cross-dot system will be useful for estimating the cariogenic potential of S. mutans isolates.

We previously isolated a variant strain, Xc100L, which shows decreased production of a surface protein antigen with a molecular mass of 190 kDa (PAc), after repeated subculturing of Streptococcus mutans strain Xc [Koga, T. et al. (1989) J.Gen. Microbiol. 135, 3199-3202]. In the present study, the levels of expression of the gtfB, gtfC, gtfD and ftf genes coding for polysaccharide-synthesizing enzymes in strain Xc100L were compared with those in strain Xc. Western blot analysis revealed multiple differences in the levels of production of these enzymes between these two strains. The amounts of the gtfB and gtfC gene products responsible for water-insoluble glucan synthesis in strain Xc100L were lower than those in strain Xc, whereas the amounts of the gtfD and ftf gene products responsible for water soluble glucan synthesis and fructan synthesis, respectively, in strain Xc100L were higher than those in strain Xc. Northern blot analysis revealed that the amounts of the four enzymes and PAc produced by strain Xc100L reflected the relative amounts of mRNAs from the genes. The chloramphenicol acetyltransferase gene was fused with each of these five genes, and the transcriptional activity of each gene in strain Xc100L was quantitatively compared with that in strain Xc. The chloramphenicol acetyltransferase assay also indicated that the phenotypic differences between strain Xc and strain Xc100L were due to differences in the transcriptional activities of the virulence genes. No differences in the nucleotide sequences of the promoter regions of the gtfB, gtfC, gtfD, ftf and pac genes were found between strain Xc and strain Xc100L. It is possible that a factor(s) affecting the levels of transcription of the multiple virulence genes exists in S. mutans.

Four putative open reading frames (ORFs) were previously identified in the regions flanking the Streptococcus mutans GS-5 fructosyltransferase (FTF) gene. One of these, ORF 3, appeared to code for a low-molecular-mass protein containing amino acid sequences sharing homology with several Gram-positive bacterial DNA-binding proteins and it was suggested that the ORF 3 gene product might be an FTF regulatory protein (FRP). In order to characterize this protein, we have purified the biotinylated tag-FRP fusion protein using the PinPoint protein purification system and this fusion protein was used in gel shift assays with DNA fragments containing the ftf promoter region. FRP bound specifically to the upstream region of the ftf promoter containing the inverted repeat structure that is present upstream of the -35 sequence. In contrast, FRP did not bind to DNA fragments lacking the inverted repeat structure. The results of these experiments suggest that FRP interacts with the inverted repeat region upstream of the ftf promoter and such interactions may regulate FTF expression.

To discover the molecular properties of two distinct NADH oxidases, corresponding to H2O2-forming oxidase (NOX-1) and H2O-forming oxidase (NOX-2) induced in Streptococcus mutans, for the first step we had cloned and sequenced the nox-1 gene encoding NOX-1. In this paper, a nox-2 gene encoding NOX-2 from S. mutans was cloned, and the nucleotides sequenced. The nox-2 gene comprises 1371 base-pairs, encoding a polypeptide of 457 amino acid residues. The deduced relative molecular mass (M(r) = 49919) agreed with the previous value obtained from the purified NOX-2 protein. The nox-2 gene was expressed in Escherichia coli using its own promoter. Alignment of the NOX-2 protein sequence with that of the NOX-1 showed that the proteins do not significantly resemble each other. Comparisons with the NADH oxidase from Streptococcus faecalis 10C1 yield identities of 41%. The redox-active cysteine in the enzyme from S. faecalis was found to correspond to Cys 44 in the NOX-2.

Novel cyclic isomaltooligosaccharides, cyclodextran, strongly inhibited the dextransucrase reaction. The inhibition was dependent on the cyclodextran concentration and greatly enhanced by the first incubation at 30 degrees for 30 min. Cyclodextran-heptaose and -octaose were competitive inhibitors for sucrose yielding Ki's of 0.25 and 0.64 mM, respectively. Both reducing sugar and dextran producing activities of dextransucrase were almost equally inhibited by the cyclodextrans. Although gamma-cyclodextrin, palatinose, sucrose-monocaprate, and maltitol gave 5-35% inhibition, cyclodextran-heptaose gave 95% inhibition. Moreover, water-insoluble glucan (mutan) synthesis by the glucosyltransferase from Streptococcus mutans was significantly repressed by the addition of cyclodextran.

We have constructed a panel of mutants of S. mutans V403 which are defective in one or more of the glucosyltransferase genes (gtfB, C or D) or the fructosyltransferase gene (ftf). These strains have been tested for virulence in a gnotobiotic rat caries model with reference to both buccal (smooth surface) and sulcal (pit and fissure) carious lesions. Our data suggest differing roles for degradable and non-degradable polymers at buccal and sulcal sites. Non-degradable polymers (made by products of the gtfB and C genes) contributed significantly to the severity of smooth surface lesions. However, our studies suggested their role in pit and fissure lesions was not as important as the role of degradable polymers. Specifically, a mutant deficient in the major insoluble glucan synthesizing activity (product of the gtfB gene) was 25% more cariogenic on sulcal surfaces than was the wild-type V403 strain. We propose that extracellular glucosyltransferases and fructosyltransferase compete for sucrose and that this competition influences pathogenicity at differing tooth sites.

The gene encoding L-lactate dehydrogenase (LDH) was cloned from an industrial dairy strain of Streptococcus thermophilus M-192 using a synthetic oligonucleotide probe based on the N-terminal amino acid sequence of the purified enzyme, and its nucleotide sequence was determined. The enzyme was deduced to have 328 amino acid residues with a molecular weight of 35,428 and found to have high sequence similarity to LDHs from other lactic acid bacteria (89.0% to Streptococcus mutans, 76.3% to Lactococcus lactis subsp. lactis, 67% to Lactobacillus casei, and 60% to Lactobacillus plantarum). The gene contained a promoter-like sequence similar to the Escherichia coli promoter consensus, and expression of the S. thermophilus LDH gene was observed in E. coli cells.

Fluoride at concentrations greater than 0.01 mM was found to be a quasi-irreversible inhibitor of enolase of permeabilized cells of Streptococcus mutans GS-5 and also of isolated yeast enolase. The inhibition appeared to be of the type that has been described for P-ATPases, but was not dependent on added Al3+ or Be2+ ions. Fluoride inhibition of enolase was not reversed by repeatedly washing the permeabilized cells in chilled fluoride-free medium but could be reversed by the product, phosphoenolpyruvate, or by very high levels of the substrate, 2-phosphoglycerate. Irreversible inhibition of glycolysis was not evident after fluoride treatment of intact cells, washing to remove unbound or loosely bound fluoride and addition of glucose, presumably because intracellular levels of phosphoenolpyruvate were sufficiently high to preclude irreversible fluoride inhibition of enolase.

A gene encoding a phosphomannose isomerase from Streptococcus mutans GS-5 was identified immediately downstream from the fructokinase gene, scrK. Nucleotide sequence analysis of this region revealed an open reading frame (ORF) specifying a putative protein of 316 amino acids. The gene cloned in Escherichia coli expressed strong phosphomannose isomerase activity. The deduced amino acid sequence of the pmi gene has no significant similarity with any of the previously reported phosphomannose isomerase enzymes. Insertional inactivation of the upstream gene, scrK, in S. mutans also drastically reduced phosphomannose isomerase activity and the ability of the organism to utilize mannose as a sole carbon source. These results suggest that the S. mutans pmi gene constitutes an operon with the scrK gene.

The extracellular glucosyltransferases (GTFs) of Streptococcus mutans are not secreted into the periplasmic space of Escherichia coli when the corresponding gtf genes are isolated in the latter organism. The utilization of both deletion analysis and gtfB: phoA fusions indicate that the signal sequences of the GTFs are functional in E. coli. However, these results further suggest that amino acid sequences present in the carboxyl terminus of the GTFs inhibit secretion through the cytoplasmic membrane in E. coli.

The expression of Streptococcus mutans mutant glucosyltransferase-I enzymes in S. sanguis and S. milleri suggests that cell-associated glucosyltransferase activity is dependent upon both glucan synthesis and glucan binding by the carboxyl-terminal repeating units of the enzyme. Mutant enzymes lacking these repeating units were only present in the extracellular fluids of these transformed streptococcal strains.

Sequential deletion of the carboxyl-terminal amino acids (including the six direct repeating units) of the glucosyltransferase-I (GTF-I) enzyme of Streptococcus mutans revealed differential effects on sucrase and GTF activities. Removal of all but one repeating unit resulted in a truncated enzyme with significant sucrase activity but no detectable GTF activity. These results are compatible with the presence of two functional domains in the enzyme.

Parotid salivas of 97 young adults were screened for IgA antibody to glucosyltransferase (GTF) from laboratory strains of Streptococcus mutans (serotypes c and g). Antibody levels to GTF from serotype c positively correlated with levels to serotype g GTF among these salivas. GTF's were prepared from S. mutans obtained from a subset of individuals in this population. All but one saliva showed IgA antibody activity to all of the GTF tested. In addition, the relative magnitude of each subject's antibody level was generally the highest to the GTF from their own S. mutans. Fractions, enriched for IgA by ammonium sulphate precipitation and gel filtration, showed patterns of functional inhibition of GTF activity which were consistent with patterns of IgA antibody activity in ELISA of unfractionated salivas. These data indicate that detectable levels of IgA antibody to S. mutans GTF exist in many young adult salivas, while this IgA antibody activity reacts with GTF from different biotypes, subjects generally show the highest secretory IgA antibody levels to their own GTF, and the relative amount of IgA antibody to GTF and the ability to inhibit GTF activity are roughly correlated.